María López-Bravo

CCL2 Shapes Macrophage Polarization by GM-CSF and M-CSF: Identification of CCL2/CCR2-Dependent Gene Expression Profile

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF– and M-CSF–polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF–polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF–polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow–derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.

In Vivo Induction of Immune Responses to Pathogens by Conventional Dendritic Cells

Specific defense mechanisms against pathogens are fulfilled by different subsets of nonmucosal conventional dendritic cells (DCs), including migratory Langerhans cells (LCs), dermal DCs, and resident CD8(+) and CD8(-) DCs found in lymphoid organs. Dermal DCs capture antigens in the skin and migrate to lymph nodes, where they can transfer the antigens to CD8(+) DCs and activate CD4(+) T cells. Differential antigen-processing machinery grants CD8(+) DCs a high efficiency in activating CD8(+) T cells through crosspresentation, whereas CD8(-) DCs preferentially trigger CD4(+) T cell responses. Recent findings have revealed the important role played by monocyte-derived DCs (mo-DCs), newly formed during infection, in activating CD4(+) and CD8(+) T cells, regulating immunoglobulin production, and killing pathogens. However, a number of controversial issues regarding the function of different DC subsets during viral, bacterial, and parasitic infections remain to be resolved.

Imaging of plasmacytoid dendritic cell interactions with T cells

Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4(+) T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC-T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

Prion Protein Expression by Mouse Dendritic Cells Is Restricted to the Nonplasmacytoid Subsets and Correlates with the Maturation State

Expression of the physiological cellular prion protein (PrPC) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrPC, which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrPC. Highest levels of both PrPC and MHC class II molecules were displayed by lymph node CD8αint DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrPC, as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220− DCs confirmed that PrPC expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220+ plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrPC, both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8α+ DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrPC expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.

RUNX3 negatively regulates CD36 expression in myeloid cell lines.

CD36 is a member of the scavenger receptor type B family implicated in the binding of lipoproteins, phosphatidylserine, thrombospondin-1, and the uptake of long-chain fatty acids. On mononuclear phagocytes, recognition of apoptotic cells by CD36 contributes to peripheral tolerance and prevention of autoimmunity by impairing dendritic cell (DC) maturation. Besides, CD36 acts as a coreceptor with TLR2/6 for sensing microbial diacylglycerides, and its deficiency leads to increased susceptibility to Staphylococcus aureus infections. The RUNX3 transcription factor participates in reprogramming DC transcription after pathogen recognition, and its defective expression leads to abnormally accelerated DC maturation. We present evidence that CD36 expression is negatively regulated by the RUNX3 transcription factor during myeloid cell differentiation and activation. In molecular terms, RUNX3 impairs the activity of the proximal regulatory region of the CD36 gene in myeloid cells through in vitro recognition of two functional RUNX-binding elements. Moreover, RUNX3 occupies the CD36 gene proximal regulatory region in vivo, and its overexpression in myeloid cells results in drastically diminished CD36 expression. The down-regulation of CD36 expression by RUNX3 implies that this transcription factor could impair harmful autoimmune responses by contributing to the loss of pathogen- and apoptotic cell-recognition capabilities by mature DCs.

Statins inhibit iNOS-mediated microbicidal potential of activated monocyte-derived dendritic cells by an IFN-β-dependent mechanism.

Statins are prescribed to 25 million people worldwide for treating hypercholesterolemia and reducing the risk of cardiovascular diseases. However, the side effects of statins on immunity, and particularly on DC immunobiology, have not been analyzed in‐depth. Here, we have investigated the impact of lovastatin treatment during monocyte differentiation into DCs on the responsiveness of the resulting monocyte‐derived DCs (moDCs) to TLR‐mediated activation. Lovastatin positively regulated TLR4 signaling in LPS‐stimulated moDCs, leading to strong activation of p38 MAP‐kinase paralleled by increased proinflammatory cytokine and IFN‐β production. In contrast, lovastatin promoted negative regulation of IFN‐β‐mediated autocrine signaling through the IFN‐αβ receptor, paralleled by low expression of the transcription factor IRF‐1, leading to the inhibition of the enzymes iNOS and HO‐1. Defective activation of iNOS/HO‐1 resulted in limited cytoprotective capacity against ROS and reduced microbicidal potential. These data were validated using an in vivo model of Listeria monocytogenes infection, which revealed that iNOS activation by splenic inflammatory moDCs, specialized in NO and TNF‐α production, was strongly reduced in lovastatin‐treated, Listeria‐infected mice. Statin treatment could have severe implications in immunity against pathogens due to defective iNOS/HO‐1 metabolism activation in inflammatory moDCs that might lead to immune failure.

Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation

Summary Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6Chigh monocytes in NK cell‐mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti‐Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon‐dependent IL‐15 production by spleen inflammatory Ly6Chigh monocytes to drive efficient activation and GM‐CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL‐15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL‐15‐based antifungal immunotherapies. Graphical Abstract Figure. No Caption available. HighlightsNK cell and neutrophil activation during C. albicans infection requires IL‐15Inflammatory Ly6Chigh monocytes are the main source of IL‐15 in response to C. albicans infectionType I IFN controls C. albicans‐induced IL‐15 production by inflammatory monocytesDefense against systemic candidiasis requires spleen‐kidney cooperative immunity &NA; The kidney is the main target organ in systemic C. albicans infection. Domínguez‐Andrés et al. now show that effective defense against systemic candidiasis relies on type I interferon‐dependent IL‐15 production by spleen inflammatory monocytes, which drives splenic NK cell activation and GM‐CSF release that in turn boost the candidacidal potential of kidney neutrophils.

IL-4 blocks TH1-polarizing/inflammatory cytokine gene expression during monocyte-derived dendritic cell differentiation through histone hypoacetylation

BACKGROUND Whereas recent research has characterized the mechanism by which dendritic cells (DCs) induce T(H)1/T(H)17 responses, the functional specialization enabling DCs to polarize T(H)2 responses remains undefined. Because IL-4 is essential during T(H)2 responses not only by acting on CD4(+) T cells through the activation of GATA-3 but also by regulating IgE class-switching, epithelial cell permeability, and muscle contractility, we hypothesized that IL-4 could also have a role in the conditioning of DCs during T(H)2 responses. OBJECTIVE We sought to analyze whether IL-4 exerts an immunomodulatory function on DCs during their differentiation, leading to their functional specialization for the induction of T(H)2 responses. METHODS Monocyte-derived DCs (moDCs) conditioned by IL-4 during their differentiation (IL-4-conditioned moDCs [IL-4-moDCs]) were analyzed for T(H)1-polarizing/inflammatory cytokine production in response to Toll-like receptor stimulation. The acetylation level of the promoters of the genes encoding these cytokines was analyzed by using chromatin immunoprecipitation. Gene expression profiling of IL-4-moDCs was defined by using mouse genome microarrays. IL-4-moDCs were tested for their capacity to induce house dust mite-mediated allergic reactions. RESULTS Our data suggest that IL-4 inhibits T(H)1-polarizing/inflammatory cytokine gene expression on IL-4-moDCs through the deacetylation of the promoters of these genes, leading to their transcriptional repression. Microarray analyses confirmed that IL-4 upregulated T(H)2-related genes as eosinophil-associated ribonucleases, eosinophil/basophil chemokines, and M2 genes. IL-4 licensed moDCs for the induction of T(H)2 responses, causing house dust mite-mediated allergic airway inflammation. CONCLUSION This study describes a new role for IL-4 by demonstrating that moDCs are conditioned by IL-4 for the induction of T(H)2 responses by blocking T(H)1-polarizing/inflammatory cytokine production through histone hypoacetylation and upregulating T(H)2-related genes.

IVIg Promote Cross-Tolerance against Inflammatory Stimuli In Vitro and In Vivo

IVIg is an approved therapy for immunodeficiency and for several autoimmune and inflammatory diseases. However, the molecular basis for the IVIg anti-inflammatory activity remains to be fully explained and cannot be extrapolated from studies on animal models of disease. We now report that IVIg impairs the generation of human monocyte–derived anti-inflammatory macrophages by inducing JNK activation and activin A production and limits proinflammatory macrophage differentiation by inhibiting GM-CSF–driven STAT5 activation. In vivo, IVIg provokes a rapid increase in peripheral blood activin A, CCL2, and IL-6 levels, an effect that can be recapitulated in vitro on human monocytes. On differentiating monocytes, IVIg promotes the acquisition of altered transcriptional and cytokine profiles, reduces TLR expression and signaling, and upregulates negative regulators of TLR-initiated intracellular signaling. In line with these effects, in vivo IVIg infusion induces a state tolerant toward subsequent stimuli that results in reduced inflammatory cytokine production after LPS challenge in human peripheral blood and significant protection from LPS-induced death in mice. Therefore, IVIg conditions human macrophages toward the acquisition of a state of cross-tolerance against inflammatory stimuli, an effect that correlates with the net anti-inflammatory action of IVIg in vivo.